Extended Data Fig. 2: Shh-induced Ang1 is required for OPC specification.
a, Scheme showing the steps to obtain neural progenitor-derived neurospheres (NSPs) from a single-cell suspension collected from E11.5 SCs via ‘open book preparation’. After the second passage NSPs were stimulated for RNA extraction. b, Image of Secondary NSPs (NSP-2) used for all experiments. Data is representative from at least 3 independent experiments. Scale bar 100μm. c,d, NSPs (in first (NSP-1) or second passage (NSP-2)) express characteristic neural stem cell markers such as Sox2 and Nestin (c). NSPs express components of the Shh pathway (Shh, Ptch and Gli1), Ang1, but not its receptor Tie2, its co-receptor Tie1 nor the endothelial cell marker Cd31 (d). Data is representative from 3 independent experiments. e, Scheme illustrating the steps for preparation of flat-mount SC explants. The SC of E11.5 embryos was isolated by ‘open book preparation’. SC ‘open books’ were cultured for 24 h. The dashed line indicates the limit of the ventricular zone (VZ) and the mantle zone (MZ). V: ventral; D: dorsal; A: anterior; P: posterior. FP: floor plate. pMN: motor neuron progenitor domain. f, Example of 3D rendering of explants images (images are shown in 2D in main Fig. 1e). Scale bar 50μm. g, Transverse sections of wild-type SCs explants immunostained for Olig2 and Sox10 to detect OPCs in the VZ (open arrowheads) and MZ (filled arrowheads) after culturing with vehicle (control DMSO), Shh inhibitor Sant1 300 nM, and/or Ang1 300 ng/mL for 24 hours. Scale bar 100 µm. h,i, Relative expression of Ptch (h) and Ang1 (i) mRNA of control (vehicle) and Sant1 treated wild-type SC explants. Graphs show mean±SEM (Control n = 16, Sant1 n = 15 of 4 independent litters). Unpaired two-sided T-Test, Ptch ****p<0.0001, Ang1**p=0.0062. j, Quantification of the number of OPCs in the MZ of the different conditions shown in (g), normalized to control. Graph show mean±SEM (Control n = 12, Sant1 n = 11, Sant1+Ang1 n = 12, of 5 independent litters). One-way ANOVA, Control vs +Sant1 **p=0.0027; +Sant1 vs +Sant1+Ang1 *p=0.0155.
